This study was undertaken to determine pattern sensitivity of phrenic nerve plasticity in respect to different respiratory challenges and to investigate the role of 5-HT and α2-adrenergic receptors in the initiation of pLTD. Study was divided into two parts. In the first, we compared long-term effects of intermittent and continuous hypercapnic and hypoxic stimuli, and combined intermittent hypoxia and hypercapnia on phrenic nerve plasticity. In the second part, different drugs were microinjected in the raphe region to investigate which receptors are important for pLTD induction and maintance. Adult, male, urethane-anesthetized, vagotomized, paralyzed, mechanically ventilated Sprague-Dawley rats were exposed to: acute intermittent hypercapnia (AIHcair or AIHcO2), acute intermittent hypoxia (AIH), combined intermittent hypercapnia and hypoxia (AIHcH), continuous hypercapnia (CHc), or continuous hypoxia (CH). In the second part of the study rats were exposed to the protocol of acute intermittent hypercapnia (5 episodes of 15% CO2 in air, each episode lasting 3 minutes). Experimental group received microinjection of selective 5-HT1A receptors agonist 8-OH-DPAT, broad spectrum 5-HT antagonist methysergide or α2- adrenergic antagonist yohimbine, whereas control group received microinjection of 0.9% saline into the caudal raphe region. Peak phrenic nerve activity (pPNA) and burst frequency were analyzed during baseline (T0), hypercapnia or hypoxia exposures, at 15, 30, and 60 minutes (T60) after the end of the stimulus. In the AIHc groups phrenic nerve frequency decreased from 44.25 ± 4.06 at T0 to 35.29 ± 5.21 breaths/min at T60, (P=0.04, AIHcair) and from 45.5 ± 2.62 to 37.17 ± 3.68 breaths/min (P=0.05, AIHcO2), i.e. frequency phrenic long term depression was induced. Exposure to AIH elicited increase of pPNA at T60 by 143.7 ± 30.1 % compared to baseline (P=0.02), i.e. phrenic long-term facilitation was induced. Combined intermittent hypercapnia and hypoxia or continuous hypercapnia or hypoxia failed to induce long-term plasticity of the phrenic nerve. In the second part of the study, in the control group, pPNA decreased 60 minutes after the end of the last hypercapnic episode compared to baseline values, i.e. pLTD developed (P=0.02). In the 8-OH-DPAT group pPNA significantly decreased at T15, T30 and T60 compared to baseline, i.e. pLTD developed (P=0.01). In the methysergide-treated group, acute intermittent hypercapnia did not evoke significant changes in pPNA at T15, T30, and T60 compared to baseline values. In the yohimbine group acute intermittent hypercapnia did not evoke significant changes of the peak phrenic nerve activity at T15, T30, and T60 compared to baseline values after the end of the last hypercapnic episode. In conclusion, intermittency of the hypercapnic or hypoxic stimuli is needed to evoke phrenic nerve plasticity. Activation of 5-HT1A receptors accentuated induction of pLTD, whereas blockade of α2-adrenergic receptors prevented development of pLTD following acute intermittent hypercapnia in anesthetized rats. These results suggest that chemical modulation of 5-HT and α2-adrenergic receptors in raphe nuclei affects hypercapnia induced pLTD, offering the important insights in understanding the mechanisms involved in development of respiratory plasticity.